[unreadable] Studies of the molecular basis of Alzheimer's disease (AD) exemplify the increasingly blurred distinction between basic and applied biomedical research. Genetic, biochemical, cell biological and animal modeling studies all support the hypothesis that accumulation of amyloid beta-protein (AB) causes AD, but it is not known why cerebral AB levels are elevated in the vast majority of AD patients. While great attention has been focused on the mechanisms of AB production, only a small number of cases is known to involve heightened production. Therefore, many cases of typical (late-onset) AD could be caused by faulty clearance of a peptide that is made at normal levels throughout life. In this competing renewal application, we wish to continue to pursue two hypotheses: (a) that defects in proteases which normally degrade AB may underlie some or many cases of familial and "sporadic" late-onset AD; and (b) that whether or not this turns out to be true, subtly activating or disinhibiting such proteases could lower brain AB levels therapeutically. Addressing this previously understudied topic requires a thorough understanding of which proteases regulate steady-state AB levels in vivo and how they do so. To this end, we propose four interrelated Specific Aims: 1. To test whether chronic dysfunction of insulin-degrading enzyme ODE) leads to decreased AB degradation and cerebral AB accumulation in vivo in two compelling models: the GK rat, which develops type II diabetes caused by missense mutations in IDE, and a newly generated IDE knock-out mouse; 2. To quantify the relative roles of the 3 best characterized AB-degrading proteases -- neprilysin, IDE and uPA/plasminogen -- in cerebral AB economy by comparing their effects in the progeny of the relevant transgenic and knock-out mice bred to APP transgenic mice; 3. To elucidate the normal cell biology and membrane trafficking of lDE that enables it to mediate the degradation of both extracellular/intraluminal substrates (e.g., insulin, amylin, AB) and cytosolic substrates (e.g., the APP intracellular domain); and 4. To use a chemical biology approach to identify small-molecule activators and inhibitors of IDE and neprilysin as mechanistic and therapeutic tools. The proposed experiments are based on extensive preliminary data, including the establishment of the necessary rodent models, and will allow us to extend a new line of investigation in the AD field that has emanated in part from this grant. We believe the results could have broad implications for the pathogenesis and treatment of AD as well as for peptide turnover in the brain and the fundamental cell biology of proteases. [unreadable] [unreadable]